TCR Mimic Antibodies as Vascular Targeting Tools

ABSTRACT

The present invention includes a method of delivering a therapeutic agent into and across an endothelial cell (EC) in a subject in need thereof, comprising: identifying a subject in need for treatment of a cancer in the brain; attaching to a TCR mimic an active agent to form a therapeutic agent; and administering to the subject the therapeutic agent in a pharmaceutically acceptable carrier, wherein the therapeutic agent effectively crosses the blood-central nervous system microvascular barrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and is a continuation-in-partapplication of, U.S. Ser. No. 14/007,164, filed Sep. 24, 2013, whichclaims priority to and is the National Stage of InternationalApplication No. PCT/US2012/030406 filed on Mar. 23, 2012, and claimspriority to U.S. Provisional Patent Application Ser. No. 61/467,215,filed on Mar. 24, 2011, the contents of which are incorporated byreference herein in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support by the DOD grantnumber BC 111520. The government has certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of cell targeting,and more particularly, to T cell receptor mimics that target peptide-MHCcomplexes.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with T Cell Receptor mimics.

The present inventors have previously demonstrated Antibodies as T cellreceptor mimics, methods of production and uses for the same. Forexample, U.S. Patent Application No. 20090304679, filed by Weidanzteaches a methodology of producing and utilizing antibodies thatrecognize peptides associated with a tumorigenic or disease state,wherein the peptides are displayed in the context of HLA molecules.These antibodies may be utilized in therapeutic methods of mediatingcell lysis.

The inventors have previously taught that Class I majorhistocompatibility complex (MHC) molecules, designated HLA class I inhumans, bind and display peptide antigen ligands upon the cell surface.The peptide antigen ligands presented by the class I MHC molecule arederived from either normal endogenous proteins (“self”) or foreignproteins (“non-self”) introduced into the cell. Non-self proteins may beproducts of malignant transformation or intracellular pathogens such asviruses. In this manner, class I MHC molecules convey informationregarding the internal milieu of a cell to immune effector cellsincluding but not limited to, CD8.sup.+ cytotoxic T lymphocytes (CTLs),which are activated upon interaction with “non-self” peptides, therebylysing or killing the cell presenting such “non-self” peptides.

Class II MHC molecules, designated HLA class II in humans, also bind anddisplay peptide antigen ligands upon the cell surface. Unlike class IMHC molecules which are expressed on virtually all nucleated cells,class II MHC molecules are normally confined to specialized cells, suchas B lymphocytes, macrophages, dendritic cells, and other antigenpresenting cells which take up foreign antigens from the extracellularfluid via an endocytic pathway. The peptides they bind and present arederived from extracellular foreign antigens, such as products ofbacteria that multiply outside of cells, wherein such products includeprotein toxins secreted by the bacteria that often have deleterious andeven lethal effects on the host (e.g., human). In this manner, class IImolecules convey information regarding the fitness of the extracellularspace in the vicinity of the cell displaying the class II molecule toimmune effector cells, including but not limited to, CD4⁺ helper Tcells, thereby helping to eliminate such pathogens. The extermination ofsuch pathogens is accomplished by both helping B cells make antibodiesagainst microbes, as well as toxins produced by such microbes, and byactivating macrophages to destroy ingested microbes.

Class I and class II HLA molecules exhibit extensive polymorphismgenerated by systematic recombinatorial and point mutation events duringcell differentiation and maturation resulting from allelic diversity ofthe parents; as such, hundreds of different HLA types exist throughoutthe world's population, resulting in a large immunological diversity.Such extensive HLA diversity throughout the population is the root causeof tissue or organ transplant rejection between individuals as well asof differing individual susceptibility and/or resistance to infectiousdiseases. HLA molecules also contribute significantly to autoimmunityand cancer.

Class I MHC molecules alert the immune response to disorders within hostcells. Peptides which are derived from viral- and tumor-specificproteins within the cell are loaded into the class I molecule's antigenbinding groove in the endoplasmic reticulum of the cell and subsequentlycarried to the cell surface. Once the class I MHC molecule and itsloaded peptide ligand are on the cell surface, the class I molecule andits peptide ligand are accessible to cytotoxic T lymphocytes (CTL). CTLssurvey the peptides presented by the class I molecule and destroy thosecells harboring ligands derived from infectious or neoplastic agentswithin that cell.

The value of monoclonal antibodies which recognize peptide-MHC complexeshas been recognized by others (see for example Reiter, US PublicationNo. U.S. 2004/0191260 A1, filed Mar. 26, 2003; Andersen et al., USPublication No. U.S. 2002/0150914 A1, filed Sep. 19, 2001; Hoogenboom etal., US Publication No. U.S. 2003/0223994 A1, filed Feb. 20, 2003; andReiter et al., PCT Publication No. WO 03/068201 A2, filed Feb. 11,2003). However, these processes employ the use of phage displaylibraries that do not produce a whole, ready-to-use antibody product.The majority of these antibodies were isolated from bacteriophagelibraries as Fab fragments (Cohen et al., 2003; Held et al., 2004; andChames et al., 2000) and have not been examined for anti-tumor activitysince they do not activate innate immune mechanisms (e.g.,complement-dependent cytotoxicity [CDC]) or antibody-dependent cellularcytotoxicity (ADCC). Demonstration of anti-tumor activity is critical,as therapeutic mAbs are thought to act through several mechanisms, whichengage the innate response, including antibody or complement-mediatedphagocytosis by macrophage, CDC and ADCC (Liu et al., 2004; Prang etal., 2005; Akewanlop et al., 2001; Clynes et al., 2000; and Masui etal., 1986). These prior art methods also have not demonstratedproduction of antibodies capable of staining tumor cells in a robustmanner, implying that they are of low affinity or specificity. Theimmunogen employed in the prior art methods uses MHC which has been“enriched” for one particular peptide, and therefore such immunogencontains a pool of peptide-MHC complexes and is not loaded solely withthe peptide of interest. In addition, there has not been a concertedeffort in these prior art methods to maintain the structure of the threedimensional epitope formed by the peptide/HLA complex, which isessential for generation of the appropriate antibody response. For thesereasons, immunization protocols presented in these prior art referenceshad to be carried out over long periods of time (i.e., approximately 5months or longer).

In addition, the vast majority of phage-derived antibodies produced bythe prior art methods will not fold right in mammalian cells due totheir selection for expression in prokaryotic or simple eukaryoticsystems; generally, <1% of phage-derived antibodies will efficientlyfold in mammalian cells, thus greatly increasing the number ofcandidates that must be screened and virtually assuring that interestinglead candidates with the most desirable binding properties arenon-producible in mammalian cells due to the infrequency of success.Supporting this contention is the fact that very few phage-derivedantibodies have proceeded into clinical investigation, and nophage-derived antibody has been approved for use as a therapeutic. Allapproved therapeutic antibodies have their discovery origin from amammalian species.

Thus, the prior art phage-derived antibodies are not useful for makinganti-MHC/peptide complexes, as they typically exhibit low affinity, lowrobustness, low capability to grow and fold, and as they are generallylaborious to implement and have not been shown to be viable for approvedtherapeutic use.

SUMMARY OF THE INVENTION

The present inventors recognized that a need exists in the art fortherapeutic antibodies with novel recognition specificity forpeptide-HLA domain in complexes present on the surface of epithelialcells throughout the body. The presently claimed and disclosed inventionprovides innovative processes for using antibody molecules endowed withunique antigen recognition specificities for peptide-HLA complexes, aspeptide-HLA molecules, to not only enter endothelial cells at theblood-brain-barrier (BBB), but trancytose past the BBB into protectedneural tissues.

In one embodiment, the present invention includes a method of deliveringa therapeutic agent into and across an endothelial cell (EC) in asubject in need thereof, comprising: identifying a subject in need fortreatment of a cancer in the brain; attaching to a TCR mimic an activeagent to form a therapeutic agent; and administering to the subject thetherapeutic agent in a pharmaceutically acceptable carrier, wherein thetherapeutic agent effectively crosses the blood-central nervous systemmicrovascular barrier. In one aspect, the barrier being crossed is theblood-brain barrier (BBB), blood-retina barrier, blood-nerve barrier orblood-spinal cord barrier. In another aspect, the disease or disorder isa cancer other than a brain cancer that has metastasized to the brain.In another aspect, the disease or disorder is a breast cancer that hasmetastasized to the brain. In another aspect, the TCR mimic is anantibody or fragment thereof and is selected from RL6A or RL21A. Inanother aspect, the therapeutic agent or carrier is directly conjugatedto a targeting molecule by: (a) non-specific or specific protein-proteininteraction; (b) covalent bonding; (c) non-covalent bonding; or (d)coordinating chemical bonding; which conjugation is optionally effectedvia a spacer or linker that bridges between the therapeutic agent orcarrier and the targeting molecule, or (e) a recombinant fusion orhybrid polypeptide. In another aspect, the step of administering is (a)by continuous intravenous or intraarterial infusion; or (b) by bolusinjection by an intravenous, intramuscular, intraarterial, orintralesional route. In another aspect, the active agent is anantineoplastic agent, a cytotoxic agent, anti-inflammatory, a hormone,an enzyme, a neurotransmitter, a neurotrophic factor, antibiotics, acytokine, or a neuropeptide.

Yet another embodiment of the present invention includes a method ofdelivering a therapeutic agent into and across a microvascularblood-central nervous system (CNS) barrier in a subject, comprising:identifying a subject in need for treatment of a cancer in the brain;and administering to a subject with at least one of a CNS disease ordisorder or a peripheral disease or disorder with CNS involvement, acomposition comprising: a TCR mimic and active agent that together forma therapeutic agent; and a pharmaceutically acceptable carrier, whereinthe therapeutic agent is encapsulated in a nanocontainer to which islinked the targeting molecule, wherein an effective barrier-entering andbarrier crossing amount of the therapeutic agent enters and crosses themicrovascular blood CNS barrier. In one aspect, barrier being crossed isthe blood-brain barrier (BBB), blood-retina barrier, blood-nerve barrieror blood-spinal cord barrier. In another aspect, the disease or disorderis a cancer other than a brain cancer that has metastasized to thebrain. In another aspect, the disease or disorder is a breast cancerthat has metastasized to the brain.

In another aspect, the TCR mimic is an antibody or fragment thereof andis selected from RL6A or RL21A. In another aspect, the therapeutic agentor carrier is directly conjugated to a targeting molecule by: (a)non-specific or specific protein-protein interaction; (b) covalentbonding; (c) non-covalent bonding; or (d) coordinating chemical bonding;which conjugation is optionally effected via a spacer or linker thatbridges between the therapeutic agent or carrier and the targetingmolecule, or (e) a recombinant fusion or hybrid polypeptide. In anotheraspect, the step of administering is at least one of: (a) by continuousintravenous or intraarterial infusion; or (b) by bolus injection by anintravenous, intramuscular, intraarterial, or intralesional route. Inanother aspect, the active agent is an antineoplastic agent, a cytotoxicagent, anti-inflammatory, a hormone, an enzyme, a neurotransmitter, aneurotrophic factor, antibiotics, a cytokine, or a neuropeptide.

Yet another embodiment of the present invention includes a method fortreating a neuronal disease or condition comprising: identifying asubject in need of such treatment for a cancer in the brain;administering to the subject in need of such treatment a neuroprotectiveand/or neurorestorative agent in an amount effective to treat thedisorder in the subject, wherein the agent comprises: a TCR mimic thattargets an MHC-peptide combination found on the surface of anendothelial cells of the neuronal vasculature that is conjugated to anactive agent to form a therapeutic agent and that crosses at least oneof the blood-brain barrier, blood-retina barrier, blood-nerve barrier,or blood-spinal cord barrier. In one aspect, the disease or disorder isa cancer other than a brain cancer that has metastasized to the brain.In another aspect, the disease or disorder is a breast cancer that hasmetastasized to the brain. In another aspect, the TCR mimic is anantibody or fragment thereof and is selected from RL6A or RL21A. Inanother aspect, the therapeutic agent or carrier is directly conjugatedto a targeting molecule by: (a) non-specific or specific protein-proteininteraction; (b) covalent bonding; (c) non-covalent bonding; or (d)coordinating chemical bonding; which conjugation is optionally effectedvia a spacer or linker that bridges between the therapeutic agent orcarrier and the targeting molecule, or (e) a recombinant fusion orhybrid polypeptide. In another aspect, the step of administering is atleast one of (a) by continuous intravenous or intraarterial infusion; or(b) by bolus injection by an intravenous, intramuscular, intraarterial,or intralesional route. In another aspect, the active agent is anantineoplastic agent, a cytotoxic agent, anti-inflammatory, a hormone,an enzyme, a neurotransmitter, a neurotrophic factor, antibiotics, acytokine, or a neuropeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1 a-1 d. Binding of ¹²⁵1-labeled antibodies to hCMEC/D3 monolayers(FIG. 1 a) or HH8 monolayers (FIG. 1 b). Tracer concentrations were 2nM. (FIG. 1 c) and (FIG. 1 d) show the effect of mild acid wash onbinding to hCMEC/D3 cells. Competition by 100-fold molar excess ofunlabeled antibody is indicated as “+cold” or “+comp”.

FIGS. 2 a-2 d. Transcytosis experiments in hCMEC/D3 cells. Linearregression with 95% confidence intervals (dashed lines) was applied tothe time course of each antibody. Tracer concentrations were 2 nM,tracer+comp denotes presence of 100-fold molar excess of unlabeledantibody.

FIGS. 3 a-3 d. Transcytosis experiments in HH8 cells. Linear regressionwith 95% confidence intervals (dashed lines) was applied to the timecourse of each antibody. Tracer concentrations were 2 nM, tracer +compdenotes presence of 100-fold molar excess of unlabeled antibody.

FIGS. 4 a-4 d. Brain uptake in transgenic mouse strain 3475. (FIG. 4 a)shows Vd brain, which is the apparent volume of distribution at 60 min(=ratio of concentrations in brain and plasma) after injection of 30μg/kg tracer. Absence or presence of IFNγ pretreatment in these animalsover 48 h is indicated as +IFN or −IFN. No effect of IFNγ on Vd of UPC10was seen and both UPC10 groups were pooled to get an estimate of brainplasma volume (8.5±1.4 IJL/g, mean±SD, n=6).

FIGS. 5 a-5 d. Brain uptake in transgenic mouse strain 4191. Vd brain ofRL6A at 60 min after injection is shown in FIG. 5 a and FIG. 5 c forhemizygous (hemi) or homozygogous (homo) mice. BB7.2 data were obtainedin homozygous animals. All data in FIGS. 5 a-5 d are from homozygousmice. FIG. 5 c shows the brain concentrations calculated from (FIG. 5 a)after correction for brain plasma volume (8.5 μl/g).

FIG. 6 a Plasma kinetics in strain 3475. All mice were treated with IFNγover 48 h before tracer injection. Mean±SD, n=3-5.

FIG. 6 b Plasma kinetics in strain 4191. All mice were treated with IFNγover 48 h before tracer injection. Mean±SD, n=2-3.

FIG. 7 shows the expression of A2 Allele and YLL/HLA-A2 complex onMDA-MB-231 BR cells.

FIG. 8 shows the internalization of RL6A in MDA-MB-231 cells.

FIG. 9 shows the expression of RL6A on cell surface of brain endothelialcell line.

FIGS. 10A and 10B show the uptake of RL6A in brain endothelial cells.

FIGS. 11A and 11B show the binding of the antibodies on MDA-MB-231cells.

FIGS. 12A and 12B show the effect of the antibodies for cleaved Caspase3 (12A) and cleaved PARP (12B), with hCMEDC3 cells in growth phase andmonolayered.

FIG. 13 shows that RL6A in MDA-MB-231 BR cell line showed significantlyhigher cleaved Caspase 3 activity compared to brain endothelial cells.

FIGS. 14A and 14B show the binding of the antibodies on MDA-MB-231cells.

FIGS. 15A and 15B show the effect of the antibodies for cleaved Caspase3 (12A) and cleaved PARP (12B), with HH8 cells in growth phase andmonolayered.

FIG. 16 shows that cleaved PARP activity was significantly higher inRL6A treated MDA-MB-231 BR compared to brain endothelial cell lines.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the terms “antibody” or “antibody peptide(s)” refer toan intact antibody, or a binding fragment thereof that competes with theintact antibody for specific binding. Binding fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include Fab, Fab′, F(ab′)2, Fv, andsingle-chain antibodies. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical. An antibody substantially inhibits adhesion of a receptor toa counterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

As used herein, the term “MHC” refers to the Major HistocompabilityComplex, which is defined as a set of gene loci specifying majorhistocompatibility antigens. The term “HLA” as used herein will beunderstood to refer to Human Leukocyte Antigens, which is defined as thehistocompatibility antigens found in humans. As used herein, “HLA” isthe human form of “MHC”.

As used herein, the terms “MHC light chain” and “MHC heavy chain” referto portions of the MHC molecule. Structurally, class I molecules areheterodimers comprised of two noncovalently bound polypeptide chains, alarger “heavy” chain (a) and a smaller “light” chain (β2-microglobulinor β2 m). The polymorphic, polygenic heavy chain (45 kDa), encodedwithin the MHC on chromosome six, is subdivided into three extracellulardomains (designated 1, 2, and 3), one intracellular domain, and onetransmembrane domain. The two outermost extracellular domains, 1 and 2,together form the groove that binds antigenic peptide. Thus, interactionwith the TCR occurs at this region of the protein. The 3 domain of themolecule contains the recognition site for the CD8 protein on the CTL;this interaction serves to stabilize the contact between the T cell andthe APC. The invariant light chain (12 kDa), encoded outside the MHC onchromosome 15, consists of a single, extracellular polypeptide. Theterms “MHC light chain”, “β-2-microglobulin”, and “β2 m” may be usedinterchangeably herein.

As used herein, the term “epitope” refers to any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics. An antibody is to specificallybind an antigen when the dissociation constant is <1 μM, or <100 nM, or<10 nM.

As used herein, the term “antibody” is used in the broadest sense, andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments (e.g., Fab,F(ab′)2 and Fv) so long as they exhibit the desired biological activity.Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond. While the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Clothia et al., J. Mol.Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA82 4592-4596 (1985), relevant portions incorporated herein by reference.

As used herein, an “isolated” antibody is one that has been identifiedand separated and/or recovered from a component of the environment inwhich it was produced. Contaminant components of its productionenvironment are materials, which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In certainembodiments, the antibody will be purified as measurable by at leastthree different methods: 1) to greater than 50% by weight of antibody asdetermined by the Lowry method, such as more than 75% by weight, or morethan 85% by weight, or more than 95% by weight, or more than 99% byweight; 2) to a degree sufficient to obtain at least 10 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequentator, such as at least 15 residues of sequence; or 3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomasie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the term “antibody mutant” refers to an amino acidsequence variant of an antibody wherein one or more of the amino acidresidues have been modified. Such mutants necessarily have less than100% sequence identity or similarity with the amino acid sequence havingat least 75% amino acid sequence identity or similarity with the aminoacid sequence of either the heavy or light chain variable domain of theantibody, such as at least 80%, or at least 85%, or at least 90%, or atleast 95%.

As used herein, the term “variable” in the context of variable domain ofantibodies, refers to the fact that certain portions of the variabledomains differ extensively in sequence among antibodies and are used inthe binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) also known ashypervariable regions both in the light chain and the heavy chainvariable domains. There are at least two techniques for determiningCDRs: (1) an approach based on cross-species sequence variability (i.e.,Kabat et al., Sequences of Proteins of Immunological Interest (NationalInstitute of Health, Bethesda, Md. 1987); and (2) an approach based oncrystallographic studies of antigen-antibody complexes (Chothia, C. etal. (1989), Nature 342: 877). The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat etal.) The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector function, such asparticipation of the antibody in antibody-dependent cellular toxicity.

As used herein, the term “antibody fragment” refers to a portion of afull-length antibody, generally the antigen binding or variable region.Examples of antibody fragments include Fab, Fab′, F(ab′)2 and Fvfragments. Papain digestion of antibodies produces two identical antigenbinding fragments, called the Fab fragment, each with a single antigenbinding site, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen binding fragments which are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′). As usedherein, “functional fragment” with respect to antibodies, refers to Fv,F(ab) and F(ab′)2 fragments.

As used herein, the “Fv” fragment is the minimum antibody fragment whichcontains a complete antigen recognition and binding site. This regionconsists of a dimer of one heavy and one light chain variable domain ina tight, non-covalent association (VH-VL dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH-VL dimer.Collectively, the six CDRs confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

The Fab fragment [also designated as F(ab)] also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains have a free thiol group. F(ab′) fragments areproduced by cleavage of the disulfide bond at the hinge cysteines of theF(ab′)2 pepsin digestion product. Additional chemical couplings ofantibody fragments are known to those of ordinary skill in the art.

The light chains of antibodies (immunoglobulin) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino sequences of their constantdomain.

Depending on the amino acid sequences of the constant domain of theirheavy chains, “immunoglobulins” can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG4; IgA-1 andIgA-2. The heavy chains constant domains that correspond to thedifferent classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the presently disclosed and claimed invention may be made by thehybridoma method first described by Kohler and Milstein, Nature 256, 495(1975), relevant portions incorporated herein by reference.

All monoclonal antibodies utilized in accordance with the presentlydisclosed and claimed invention will be either (1) the result of adeliberate immunization protocol, as described in more detail hereinbelow; or (2) the result of an immune response that results in theproduction of antibodies naturally in the course of a disease or cancer.These monoclonal antibodies are distinguished from the prior artantibodies which are phage-derived, because the prior art phage-derivedantibodies are not useful for making anti-MHC/peptide complexes, as theytypically exhibit low affinity, low robustness, low capability to growand fold, and as they are generally laborious to implement and have notbeen shown to be viable for approved therapeutic use.

The uses of the monoclonal antibodies of the presently disclosed andclaimed invention may require administration of such or similarmonoclonal antibody to a subject, such as a human. However, when themonoclonal antibodies are produced in a non-human animal, such as arodent, administration of such antibodies to a human patient willnormally elicit an immune response, wherein the immune response isdirected towards the antibodies themselves. Such reactions limit theduration and effectiveness of such a therapy. In order to overcome suchproblem, the monoclonal antibodies of the presently disclosed andclaimed invention can be “humanized”, that is, the antibodies areengineered such that antigenic portions thereof are removed and likeportions of a human antibody are substituted therefore, while theantibodies' affinity for specific peptide/MHC complexes is retained.This engineering may only involve a few amino acids, or may includeentire framework regions of the antibody, leaving only thecomplementarity determining regions of the antibody intact. Severalmethods of humanizing antibodies are known in the art and are disclosedin U.S. Pat. No. 6,180,370, issued to Queen et al on Jan. 30, 2001; U.S.Pat. No. 6,054,927, issued to Brickell on Apr. 25, 2000; U.S. Pat. No.5,869,619, issued to Studnicka on Feb. 9, 1999; U.S. Pat. No. 5,861,155,issued to Lin on Jan. 19, 1999; U.S. Pat. No. 5,712,120, issued toRodriquez et al on Jan. 27, 1998; and U.S. Pat. No. 4,816,567, issued toCabilly et al on Mar. 28, 1989, relevant portions incorporated herein byreference.

Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)2 or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen etal., 1988), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, 1992).

The presently disclosed and claimed invention further includes the useof fully human monoclonal antibodies against specific peptide/MHCcomplexes. Fully human antibodies essentially relate to antibodymolecules in which the entire sequence of both the light chain and theheavy chain, including the CDRs, arise from human genes. Such antibodiesare termed “human antibodies” or “fully human antibodies” herein. Humanmonoclonal antibodies can be prepared by the trioma technique; the humanB-cell hybridoma technique (see Kozbor, et al., Hybridoma, 2:7 (1983))and the EBV hybridoma technique to produce human monoclonal antibodies(see Cole, et al., PNAS 82:859 (1985)). Human monoclonal antibodies maybe utilized in the practice of the presently disclosed and claimedinvention and may be produced by using human hybridomas (see Cote, etal., PNAS 80:2026 (1983)) or by transforming human B-cells with EpsteinBarr Virus in vitro (see Cole, et al., 1985), relevant portionsincorporated herein by reference.

In addition, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example but not by way of limitation, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inMarks et al., J Biol. Chem. 267:16007, (1992); Lonberg et al., Nature,368:856 (1994); Morrison, 1994; Fishwild et al., Nature Biotechnol.14:845 (1996); Neuberger, Nat. Biotechnol. 14:826 (1996); and Lonbergand Huszar, Int Rev Immunol. 13:65 (1995), relevant portionsincorporated herein by reference.

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO 94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. One embodiment of such a nonhumananimal is a mouse, and is termed the XENOMOUSE™ as disclosed in PCTPublication Nos. WO 96/33735 and WO 96/34096. This animal produces Bcells that secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598, issued to Kucherlapati et al. onAug. 17, 1999, and incorporated herein by reference. It can be obtainedby a method including deleting the J segment genes from at least oneendogenous heavy chain locus in an embryonic stem cell to preventrearrangement of the locus and to prevent formation of a transcript of arearranged immunoglobulin heavy chain locus, the deletion being effectedby a targeting vector containing a gene encoding a selectable marker;and producing from the embryonic stem cell a transgenic mouse whosesomatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771, issued to Hori et al.on Jun. 29, 1999, and incorporated herein by reference. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

As used herein, the term “pharmaceutical agent or drug” as used hereinrefers to a chemical compound or composition capable of inducing adesired therapeutic effect when properly administered to a patient.Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

The term patient includes human and veterinary subjects.

As used herein, a “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

As used herein, the term “Treatment” refers to both therapeutictreatment and prophylactic or preventative measures. Those in need oftreatment include those already with the disorder as well as those inwhich the disorder is to be prevented.

As used herein, the term “disorder” refers to any condition that wouldbenefit from treatment with the polypeptide. This includes chronic andacute disorders or diseases including those infectious or pathologicalconditions that predispose the mammal to the disorder in question.

As mentioned hereinabove, depending on the application and purpose, theT cell receptor mimic of the presently disclosed and claimed inventionmay be attached to any of various functional moieties. A T cell receptormimic of the presently disclosed and claimed invention attached to afunctional moiety may be referred to herein as an “immunoconjugate”. Inone embodiment, the functional moiety is a detectable moiety or atherapeutic moiety.

As is described and demonstrated in further detail hereinbelow, adetectable moiety or a therapeutic moiety may be particularly employedin applications of the presently disclosed and claimed inventioninvolving use of the T cell receptor mimic to detect the specificpeptide/MHC complex, or to kill target cells and/or damage targettissues.

The presently disclosed and claimed invention include the T cellreceptor mimics described herein attached to any of numerous types ofdetectable moieties, depending on the application and purpose. Forapplications involving detection of the specific peptide/MHC complex,the detectable moiety attached to the T cell receptor mimic may be areporter moiety that enables specific detection of the specificpeptide/MHC complex bound by the T cell receptor mimic of the presentlydisclosed and claimed invention.

While various types of reporter moieties may be utilized to detect thespecific peptide/MHC complex, depending on the application and purpose,the reporter moiety may be a fluorophore, an enzyme or a radioisotope.Specific reporter moieties that may utilized in accordance with thepresently disclosed and claimed invention include, but are not limitedto, green fluorescent protein (GFP), alkaline phosphatase (AP),peroxidase, orange fluorescent protein (OFP), β-galactosidase,fluorescein isothiocyanate (FITC), phycoerythrin, Cy-chrome, rhodamine,blue fluorescent protein (BFP), Texas red, horseradish peroxidase (HPR),and the like.

The presently disclosed and claimed invention includes the T cellreceptor mimics described herein attached to any of numerous types oftherapeutic moieties, depending on the application and purpose. Varioustypes of therapeutic moieties that may be utilized in accordance withthe presently disclosed and claimed invention include, but are notlimited to, a cytotoxic moiety, a toxic moiety, a cytokine moiety, abi-specific antibody moiety, and the like. Specific examples oftherapeutic moieties that may be utilized in accordance with thepresently disclosed and claimed invention include, but are not limitedto, small molecules, peptides, lipids, carbohydrates, nucleic acids orother molecules organic or inorganic, that are used to treat one or moreof the following conditions after crossing the blood-brain-barrier(often also described as microvascular permeability) including:neurodegenerative disorders, such as cerebrovascular accidents (CVA),Alzheimer's disease (AD), vascular-related dementia, Creutzfeldt-Jakobdisease (CJD), bovine spongiform encephalopathy (BSE), Parkinson'sdisease (PD), brain trauma, multiple sclerosis (MS), amyotrophic lateralsclerosis (ALS), Huntington's chorea; peripheral disorders with a CNScomponent, such as septic shock, hepatic encephalopathy, (diabetic)hypertension, diabetic microangiopathy, sleeping sickness, Whippledisease, and Duchenne muscular dystrophy; neuropsychiatric disorders,such as depression, autism, anxiety attention deficit hyperactivitydisorder (ADHD), neuropsychiatric systemic lupus erythematosus, bipolardisorder, schizophrenia and other psychoses; other CNS disorders, suchas brain tumors, epilepsy, migraine, narcolepsy, insomnia, chronicfatigue syndrome, mountain sickness, encephalitis, meningitis, andAIDS-related dementia.

A pharmaceutical composition of the presently disclosed and claimedinvention includes a T cell receptor mimic of the presently disclosedand claimed invention and a therapeutic moiety conjugated thereto thatspecifically targets the BBB. The pharmaceutical composition of thepresently disclosed and claimed invention may be an antineoplasticagent. A diagnostic composition of the presently disclosed and claimedinvention includes a T cell receptor mimic of the presently disclosedand claimed invention and a detectable moiety conjugated thereto.

Such therapeutic agents or active agents that can be conjugated to theTCR mimics of the present invention include, e.g., anti-tumor compounds,such as antineoplastic agents or cytotoxic drugs, such as alkylatingagents, e.g., Mechlorethamine hydrochloride (Nitrogen Mustard,Mustargen, HN2), Cyclophosphamide (Cytovan®, Endoxana®), Ifosfamide(IFEX®), Chlorambucil (Leukeran®), Melphalan (Phenylalanine Mustard,L-sarcolysin, Alkeran®, L-PAM), Busulfan (Myleran®), Thiotepa(Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine(CeeNU, CCNU), Streptozocin (Zanosar®); plant alkaloids, e.g.,Vincristine (Oncovin®), Vinblastine (Velban®, Velbe®), Paclitaxel(Taxol®), and the like; antimetabolites, e.g., methotrexate (MTX),Mercaptopurine (Purinethol®, 6-MP), Thioguanine (6-TG), Fluorouracil(5-FU), Cytarabine (Cytosar-U®®, Ara-C), Azacitidine (Mylosar®, 5-AZA)and the like; antibiotics, e.g., Dactinomycin (Actinomycin D,Cosmegen®), Doxorubicin (Adriamycin®), Daunorubicin (Daunomycin®,Cerubidine®), Idarubicin (Idamycin®), Bleomycin (Blenoxane®), Picamycin(Mithramycin®, Mithracin®), Mitomycin (Mutamycin®) and the like, andother anticellular proliferative agents, e.g., Hydroxyurea (Hydrea®),Procarbazine (Mutalane®), Dacarbazine (DTIC-Dome®), cisplatin(Platinol®), Carboplatin (Paraplatin®), Asparaginase (Elspar®),Etoposide (VePesid®, VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane(Lysodren®), Mitoxantrone (Novatrone®), and the like; gefitinib (ZD1839or Iressa®) and imatinib mesylate (Gleevec® or Glivec®); anti-cancerbiopharmaceutical drugs including antibodies (Rituxan® or rituximab;Herceptin® or trastuzumab; Zevalin® or ibritumomab tiuxetan(radiolabeled); Erbitux® or cetuximab; Avastin™ or bevacizumab orrhuMAb-VEGF) and cytokines (Intron® or α-interferon; Proleukin® IL-2 oraldesleukin) to treat primary brain tumors or brain metastasis ofsomatic tumors; anti-inflammatory drugs including antibodies (Enbrel® oretanercept; Remicade® or infliximab; Simulect® or basiliximab; Zenapax®or daclizumab; Kineret® or anakinra; Xolair® or omalizumab; Humira® oradalimumab; Antegren® or natalizumab; RhuFab™ or ranibizumab; Raptiva™or efalizumab) and cytokines such as interferon-α, interferon-β (Avonex®or interferon β-1a; Betaseron®/Betaferon® or interferon β-1b; Rebif® orinterferon-β-1a), interferon-γ interleukin 1 (IL-1), interleukin 2(IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5(IL-5), interleukin 6 (IL-6), TNF, granulocyte macrophage colonystimulating factor (GM-CSF: Leukine® or sargramostim), granulocytecolony stimulating factor (G-CSF: Neupogen® or filgrastim), macrophagecolony stimulating factor (M-CSF), platelet-derived growth factor(PDGF).

The therapeutic agents or active agents can be used to treat e.g.,neuroinflammation related to neurodegenerative disorders; neurotrophicfactors (e.g., NGF or nerve growth factor; BDNF or brain-derivedneurotrophic factor; NT3 or neurotrophin-3; NT4 or neurotrophin-4; NT5or neurotrophin-5; RDGF or retina-derived growth factor; CNTF or ciliaryneurotrophic factor; activin; bFGF or basic fibroblast growth factor;aFGF or acidic fibroblast growth factor; GDNF or glial cell line-derivedneurotrophic factor or neublastin or artemin or enovin, presephin,neurturin; CTGF or connective tissue growth factor; EGF or epithelialgrowth factor); erythropoietins (EPO) (Procrit®/Eprex® or erythropoietinalfa; Epogen® or erythropoietin; NeoRecormon® or erythropoietinβ;Aranesp® or darbepoietin alfa); growth hormone or somatotropin(Humatrope®; Protropin®/Nutropin®; Serostim®; Saizen®); anti-NogoA Mab(IN-1); NogoA antagonist of Nogo66 inhibitor (NEP1-40).

Other therapeutic agents or active agents can be used to treat, e.g.,neurodegenerative disorders; enzymes (e.g., Cerezyme® orglucocerebrosidase; Aldurazyme™ or laronidase; Aryplase™ orarylsulfatase B; 12S or iduronate-2-sulfatase; α-L-iduronidase;N-acetylgalactosamine 4-sulfatase; phenylase; aspartylglucosaminidase;acid lipase; cysteine transporter; Lamp-2; a galactosidase A; acidceramidase; α-L-fucosidase; ss-hexosaminidase A; GM2-activatordeficiency; α-D-mannosidase; ss-D-mannosidase; arylsulfatase A; saposinB; neuraminidase; α-N-acetylglucosaminidase phosphotransferase;phosphotransferase 7-subunit; heparan-N-sulfatase;α-N-acetylglucosaminidase; acetyl-CoA: N-acetyltransferase;N-acetylglucosamine 6-sulfatase; galactose 6-sulfatase; β-galactosidase;hyaluronoglucosaminidase; multiple sulfatases; palmitoyl proteinthioesterase; tripeptidyl peptidase I; acid sphingomyelinase;cholesterol trafficking; cathepsin K; α-galactosidase B; sialic acidtransporter; SOD or Cu/Zn superoxide dismutase) to treat e.g.,(neurological symptoms related to) lysosomal storage diseases or otherneurodegenerative disorders; brain-acting hormones and neurotransmitterssuch as somatostatin, oxytocin, vasopressin, guaranine, VIP,adrenocorticotropic hormone (ACTH), cholecystokinin (CCK), substance-P,bombesin, motilin, glicentin, glucagon, glucagon-like peptide (GLP-1);and neuropeptides and derivatives thereof such as peptide YY (PYY),neuropeptide Y (NPY), pancreatic polypeptide (PP), neurokinin A,neurokinin B, endorphin, enkephalin, neurotensin, neuromedin K,neuromedin L, calcitonin related peptide (CGRP), endothelin, ANP(“atrial natriuretic peptide”), BNP (“brain natriuretic peptide”), CNP(C-type natriuretic peptide”), and PACAP (“pituitary adenylate cyclaseactivating peptide”).

Other active agents can be used to treat imaging agents.

Other therapeutic agents or active agents can be neurotransmitterantagonists or agonists that do not penetrate the blood-brain barrier(such as certain NMDA receptor blockers); antibiotics, such as:aminoglycosides, e.g., amikacin, apramycin, arbekacin, bambermycins,butirosin, dibekacin, dihydrostreptomycin, fortimicin, gentamicin,isepamicin, kanamycin, micronomcin, neomycin, netilmicin, paromycin,ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin,trospectomycin; amphenicols, e.g., azidamfenicol, chloramphenicol,florfenicol, and theimaphenicol; ansamycins, e.g., rifamide, rifampin,rifamycin, rifapentine, rifaximin; β-lactams, e.g., carbacephems,carbapenems, cephalosporins, cehpamycins, monobactams, oxaphems,penicillins; lincosamides, e.g., clinamycin, lincomycin; macrolides,e.g., clarithromycin, dirthromycin, erythromycin, etc.; polypeptides,e.g., amphomycin, bacitracin, capreomycin, etc.; tetracyclines, e.g.,apicycline, chlortetracycline, clomocycline, etc.; syntheticantibacterial agents, such as 2,4-diaminopyrimidines, nitrofurans,quinolones and analogs thereof, sulfonamides, sulfones; antifungalagents, such as: polyenes, e.g., amphotericin B, candicidin,dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin,mepartricin, natamycin, nystatin, pecilocin, perimycin; syntheticantifungals, such as allylamines, e.g., butenafine, naftifine,terbinafine; imidazoles, e.g., bifonazole, butoconazole, chlordantoin,chlormidazole, etc., thiocarbamates, e.g., tolciclate, triazole, e.g.,fluconazole, itraconazole, terconazole; anthelmintics, such as:arecoline, aspidin, aspidinol, dichlorophene, embelin, kosin,napthalene, niclosamide, pelletierine, quinacrine, alantolactone,amocarzine, amoscanate, ascaridole, bephenium, bitoscanate, carbontetrachloride, carvacrol, cyclobendazole, diethylcarbamazine, etc.;antimalarials, such as: acedapsone, amodiaquin, arteether, artemether,artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata,chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine,cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine,mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid,primaquine, pyrimethamine, quinacrine, quinine, quinine, quinocide,quinine, dibasic sodium arsenate; antiprotozoal agents, such as:acranil, timidazole, ipronidazole, ethylstibamine, pentamidine,acetarsone, aminitrozole, anisomycin, nifuratel, timidazole,benzidazole, suramin.

Other therapeutic agents or active agents can be genes (includingexpression vectors and/or promoters, preferably the GFAP and/or GTPpromoters) encoding for polypeptides, proteins, peptides, enzymes,cytokines, interleukins, hormones and growth factors described hereinabove) or antisense DNA for polypeptides; and antisense probes (nucleicacids or peptide nucleic acids).

In addition to direct conjugation between the therapeutic or diagnosticmoieties and the targeting agent, such therapeutic or diagnosticmoieties may be conjugated either directly or via any of the well-knownpolymeric conjugation agents such as sphingomyelin, polyethylene glycol(PEG) or other organic polymers, and either with a single targetingagent or in combination with any of the well-known blood-brain barriertargeting moieties against the insulin, transferrin, IGF, leptin, LRP(1B) or LDL receptor on the blood-brain barrier and brain cell membrane.

The presently disclosed and claimed invention relates to methodologiesfor utilizing an agent, such as but not limited to antibodies orantibody fragments that function as T-cell receptor mimics (TCRm's),that recognize peptides displayed in the context of HLA molecules,wherein the peptide is associated with a tumorigenic, infectious,disease or immune dysfunction state at the BBB. These antibodies willmimic the specificity of a T cell receptor (TCR) such that the moleculesmay be used as therapeutic reagents. In one embodiment, the T cellreceptor mimics of the presently disclosed and claimed invention willhave a higher binding affinity than a T cell receptor. In oneembodiment, the T cell receptor mimic produced by the method of thepresently disclosed and claimed invention has a binding affinity ofabout 10 nanomolar or greater.

In one embodiment, the methods utilize a T-cell receptor mimic, asdescribed in detail hereinabove and in U.S. Ser. No. 11/809,895, filedJun. 1, 2007, and in US Published Application Nos. U.S. 2006/0034850,filed May 27, 2005, and U.S. 2007/00992530, filed Sep. 7, 2006, and20090304679 filed Feb. 27, 2009, incorporated herein by reference. TheT-cell receptor mimic utilized in the methods of the presently disclosedand claimed invention comprises an antibody or antibody fragmentreactive against a specific peptide/MHC complex, wherein the antibody orantibody fragment can differentiate the specific peptide/MHC complexfrom the MHC molecule alone, the specific peptide alone, and a complexof MHC and an irrelevant peptide. The T cell receptor mimic may beproduced by any of the methods described in detail in the patentapplications listed herein above and expressly incorporated herein byreference; for example but not by way of limitation, the T cell receptormimic may be produced by immunizing a host with an effective amount ofan immunogen comprising a multimer of two or more specific peptide/MHCcomplexes.

In one embodiment, the T cell receptor mimic may be produced by a methodthat includes identifying a peptide of interest, e.g., a peptideexpressed only in epithelial cells of the BBB or that are only expressedby those cells upon the initiation of a disease or disease condition,wherein the peptide of interest is capable of being presented by an MHCmolecule, and wherein the vaccine composition comprises the peptide ofinterest. An immunogen comprising a multimer of two or more peptide/MHCcomplexes is then formed, wherein the peptide of the peptide/MHC complexis the peptide of interest. An effective amount of the immunogen is thenadministered to a host for eliciting an immune response, wherein theimmunogen retains a three-dimensional form thereof for a period of timesufficient to elicit an immune response against the three-dimensionalpresentation of the peptide in the binding groove of the MHC molecule.Serum collected from the host is then assayed to determine if desiredantibodies that recognize a three-dimensional presentation of thepeptide in the binding groove of the MHC molecule is being produced,wherein the desired antibodies can differentiate the peptide/MHC complexfrom the MHC molecule alone, the peptide of interest alone, and acomplex of MHC and irrelevant peptide. The desired antibodies are thenisolated.

Peptides that have been utilized to produce TCRm's by the methodsdescribed in detail in U.S. Ser. No. 11/809,895, filed Jun. 1, 2007, andin US Published Application Nos. U.S. 2006/0034850, filed May 27, 2005,and U.S. 2007/00992530, filed Sep. 7, 2006, and 20090304679 filed Feb.27, 2009, are incorporated herein by reference. The use of TCRm'sproduced using any of the peptides of SEQ ID NOS: 1-97 disclosed in theabove-referenced patent applications, each of which are incorporatedherein by reference. However, it is to be understood that the presentlydisclosed and claimed invention is not limited to TCRm's produced usingthe peptides, but rather the scope of the presently disclosed andclaimed invention encompasses TCRm's raised against any specificpeptide/MHC complex.

T-cell receptor mimics (TCRm) are a novel class of monoclonal antibodieswith binding characteristics resembling T cell receptors (Weidanz etal., 2006; Weidanz et al., 2007). In particular, binding specificity isdetermined by the MHC molecule in combination with a peptide presentedin its binding groove. TCRm are currently evaluated both as diagnostictools and as therapeutics in a variety of settings, e.g. for assessmentof vaccine potency and as anti-tumor agents (Neethling et al., 2008;Verma et al., 2010; Verma et al., 2010).

The present document introduces a novel field of application for TCRm.The inventors have recently shown that TCRm bind to specific peptide-MHCtargets on human brain derived endothelial cells and undergointernalization (Bhattacharya et al., 2010). Therefore, TCRm have thepotential to be useful as vascular targeting agents. The presentinvention shows that its possible to identify peptide-MHC complexes onvascular endothelial cells of a given organ (e.g., brain), TCRm aregenerated that target such peptide-MHC complexes with high specificityand avidity. It is demonstrated herein in vitro and in vivo that theTCRm-active agent binding to a monolayer of brain-derived endothelialcells and time dependent, saturable transcytosis with a prototype TCRm,RL-6A. RL-6A recognizes a specific peptide-HLA-A2 complex derived fromp68 RNA helicase (Verma et al., 2010). Further, it is shown here with anin vivo model, transgenic mice expressing human HLA-A2, that the TCRmantibody targets brain. The endothelial cells of blood vessels in thecentral nervous system (CNS) form the blood-brain barrier (BBB), whichis currently a major obstacle to the development of drugs for diversediseases affecting the CNS, including neurodegenerative diseases (e.g.Alzheimer's Disease, Parkinson Disease), neuroinflammatory disorders(e.g. Multiple Sclerosis, ischemia/reperfusion injury associated withstroke), and primary or metastatic brain tumors. The present inventionis an efficient and specific targeting tool that allows delivery ofdiagnostic agents and active agents (e.g., drugs) to the CNS is an unmetneed and will have broad impact.

EXAMPLE 1 Demonstration of RL-6A Transcytosis Using An In Vitro Model ofthe Blood-Brain Barrier

Radiolabeling of Antibodies: Either of the monoclonal antibodies, RL6A(as described in detail hereinabove and in U.S. Ser. No. 11/809,895,filed Jun. 1, 2007, and in US Published Application Nos. U.S.2006/0034850, filed May 27, 2005, and U.S. 2007/00992530, filed Sep. 7,2006, and 20090304679 filed Feb. 27, 2009, incorporated herein byreference), BB7.2 (ATCC Accession No. HB-82) and UPC10 (isotype control1gG2a, Sigma Aldrich) was labeled with Na 125I from Perkin Elmer(Waltham, Mass., USA) using a chloramine-T method. Briefly, 1 mCi Na125I was reacted with 401Jg antibody in 5-20 μl phosphate bufferedsaline (pH 7.4), and was incubated at room temperature for 1 min 0.51μg/5 μl chloramine-Tin phosphate buffer, and following 1 min incubationwith additional 0.255 μg/2.5 μl chloramine-T. The reaction was stoppedby adding 3.2 μg/10 μ sodium metabisulfite (SigmaAldrich, St. Louis,Mo.). Labeled antibody was purified using a PD-10 SEPHADEX™ G25M gelfiltration column (GE healthcare Piscataway, N.J.), with an elution0.02M sodium phosphate buffer in 0.9% NaCl (pH 7.4). The peak fractionswere pooled and stored at −80° C. for use in the subsequent experiments.Each antibody was labeled to a specific activity of 12.5 μCi/μg (1875μCi/nmole) and a trichloroacetic acid (TCA) precipitability of >99%.

In vitro studies with endothelial cells: Two immortalized human brainderived endothelial cell lines were used: hCMEC/D3 (Weksler et al.,2005) and HH8 (Koval A, Shah, K, Abbruscato, T J, Generation of a New,Highly Restrictive, Conditionally Immortalized Human Brain MicrovascularEndothelial Cell Line. Society of Biomolecular Sciences 16th AnnualConference & Exhibition, Advancing the Science of Drug Discovery,Phoenix, Ariz., Apr. 11-15, 2010). Cells were grown on type 1 collagenpre-coated transwell filters after seeding at a density of 100,000cells/cm2. hCMEC/D3 cells are grown at 37° C. throughout, while HH8 aretemperature sensitive and grow at 33° C. After confluence the HH8monolayer is kept at 37° C. for 48 h before the binding/transportexperiments. Half of the assay media was changed after 4-7 days andtransport assays were performed within 8-10 days after seeding. INFγ (20ng/ml) was added 24 h-72 h before assay to stimulate MHC class Iexpression and processing. To qualify p68 peptide-MHC class Iexpression, both cell lines with and without addition of IFNγ werestained with RL-6A and target expression was evaluated using flowcytometric analysis.

Cell binding and transport assays: Radiolabeled antibodies 125I-RL-6A,1251-BB7.2 (pan-HLA A2), or 125I-UPC-10 at a concentration of 5×106cpm/0.5 ml (˜2 nM), in presence or absence of a >100-fold molar excessof unlabeled antibody of the same type, were added to the upper chamberof transwells and incubated with the endothelial cells at 37° C. At 15,30, 60, and 120 min, 50 μl samples from the lower chamber were withdrawnfor gamma counting and replaced with an equal volume of fresh assaymedium. At the end of the incubation, cells were washed carefully, andcell-associated radioactivity was determined by removing the membranesof the culture insert and counting it in a gamma-counter. For themeasurement of acid resistant binding, wash steps were performed withglycine buffer at pH 3.

Binding data were expressed as cpm bound per filter. Transendothelialtransport data were expressed as time-dependent volume cleared from theupper chamber. Linear regression was applied to fit the time course dataand slopes between the different antibodies were statistically comparedusing GraphPad Prism 5.0

EXAMPLE 2 Brain Uptake of RL6A In HLA-A2 Transgenic MicePharmacokinetics In HLA-A2 Transgenic Mice

Two HLA-A2 transgenic mouse strains expressing different constructs ofhuman A*0201 were obtained from Jackson labs (Bar Harbor, Me.):C57BL/6-Tg(HLA-A2.1)1 Enge/J [stock #3475]; andB6.Cg-Tg(HLA-A/H2-D)2Enge/J [stock #4191]. All animal experiments wereperformed according to NIH guidelines and were approved by theinstitutional animal care and use committee at Texas Tech UniversityHealth Sciences Center. Mice were kept under controlled temperature,light (12 h dark/12 h light cycle) and humidity and were fed standardrodent chow.

Next, 2-6 month old transgenic mice (weight range 20-25 grams) were usednaïve or after receiving intra-peritoneal (IP) injections of interferongamma (IFNγ) at a dose of 10,000 U/animal in saline two times with a 24h interval. Pharmacokinetic experiments were started 48 h after thefirst IFNγ injection. For the pharmacokinetic studies, animals were keptunder continuous anesthesia with 1-1.5% isoflurane in N2O:O2 (70:30volume %) throughout the experiments. The right common carotid arterywas catheterized with PE-10 in retrograde direction for repeated bloodsampling.

The tracers were labeled as described under “Methods” in EXAMPLE 1 andinjected i.v. into the jugular vein (70 μL in 10 mM Na-phosphate/0.15 MNaCl (pH 7.4). An aliquot (30 ul) of blood was collected atpredetermined time intervals of 1, 5, 10, 20, 30, 40 and 60 min from thePE-10 cannula inserted in the common carotid artery. For controlantibody UPC10, the pharmacokinetic sampling was done for up to 2 h. Thesample volume was replaced through the carotid cannula with salinecontaining heparin (100 U/ml). At 60 min after injection, the mice wereeuthanized and organs (brain, heart, lung, liver, kidney, spleen) werecollected and weighed. Blood samples from each time point, werecentrifuged for collection of plasma (8,600×g for 3 minutes).

Radioactivity of 5 μl aliquots of blood and plasma and of the organsamples was measured using automatic gamma counter 2470 (Perkin ElmerLife and Analytical Sciences, Waltham, Mass., USA). The radioactivityconcentration (cpm/g) was converted to percentage of injected dose (%ID/g for organs; % ID/ml for blood and plasma), and the organdistribution values were corrected for the corresponding vascular volumeof each organ using the equation,

C _(organ)=(V _(D) −V ₀)×C _(pl)(T)

Where V_(D) is the apparent organ volume of distribution at samplingtime T, V₀ is the plasma volume of the corresponding organ and C_(pl)(T)equals plasma concentration at time T. Pharmacokinetic parametersdescribing the time course of plasma or blood concentrations weredetermined by fitting concentration-time data to a biexponentialequation,

C=C ₁ e ^(−λ1t) +C ₂ e ^(−λ2t)

using nonlinear regression in Scientist 3.0 (Micromath Research, SaintLouis, Mich.). Derived parameters were calculated according to standardpharmacokinetic equations.

Brain Capillary depletion: To determine the extent of transcytosis ofthe antibodies in vivo, a capillary depletion method was used aspreviously described (Lee et al., 2000). Briefly, cerebral hemisphereswere collected from the animals after pharmacokinetic sampling for 60minutes after IV injection of ¹²⁵I MAb. The collected hemispheres wereweighed and homogenized on ice in cold physiological buffer (10 mMHEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl₂, 1 mM MgSO₄, and 10 mMD-glucose, pH 7.4) with a glass tissue grinder, followed by the additionof cold dextran solution to a final concentration of 16%. After removalof an aliquot of the homogenate, the remainder was centrifuged at4,300×g for 15 m in at 4° C. and the supernatant was carefully separatedfrom the capillary pellet. The capillary pellet was re-suspended in 0.5ml physiological buffer. Radioactivity was measured in the aliquots ofhomogenate and postvascular supernatant and in the re-suspendedcapillary pellet.

Acid Precipitation: To determine the integrity of the radiolabeledantibody, an acid precipitation assay was performed on plasma samplesobtained at each time point following IV injection of the antibody; andin brain post-vascular supernatant and capillary pellet after braincapillary depletion. Briefly, 5 μl of the plasma was added to 100 μl of2.5% BSA; precipitation was performed by additional adding 1 ml of 20%trichloroacetic acid. Similarly capillary pellets resuspended in 500 μlphysiological buffer and, 500 μl post-vascular supernatant weresubjected to TCA precipitation by adding 500 μl of 20% trichloroaceticacid. After vigorously mixing and incubating 10 min on ice, all sampleswere centrifuged at 4,000×g for 5 min at 4° C. The supernatant andprecipitate were separated and measured in a gamma counter. The resultswere expressed as the percentage of total radioactivity thatprecipitated.

Results from Example 1: The binding of radiolabeled antibodies tohCMEC/D3 and HH8 monolayers after 60 min incubation at 37° C. is shownin FIGS. 1 a and 1 b. Both cell lines bind RL-6A and BB7.2 in asaturable manner as evident from the decreased binding values in thepresence of unlabeled antibody. The binding level under competition by a100-fold molar excess of the antibody is comparable to the binding ofthe isotype control UPC-10. Further, binding of BB7.2 and RL6A wasenhanced by pretreatment of the cells with IFNγ. The relative increaseof RL-6A binding after IFNγ was similar in HH8 and hCMEC/D3 cells,although absolute binding expressed as cpm/filter, was higher inhCMEC/D3 cells. As expected for the pan HLA-A2 antibody BB7.2, itsbinding values exceeded the corresponding values for RL-6A.

The data on tracer associated with the cell monolayer following mildacid wash in FIGS. 1 a-1 d demonstrate that both RL-6A (FIG. 1 c) andBB7.2 (FIG. 1 d) were internalized by hCMEC/D3 cells in a time dependentmanner. Consistent with the total binding data (FIGS. 1 a and 1 b)tracer internalization was almost completely inhibited by competitionwith unlabeled antibody. As with total cell binding, internalizationincreased after IFNγ pretreatment of cells.

FIGS. 2 a-2 d and FIGS. 3 a-3 d depict the transendothelial transport ofantibodies from luminal (upper compartment) to basolateral (bottomcompartment). Transfer of UPC-10 tracer as 1gG_(2a) isotype controlrepresents paracellular leakage. In naïve cells (without IFNγstimulation) there were only small differences between RL-6A, BB7.2 andUPC-10. However, after IFNγ the differences increased and both RL-6A andBB7.2 clearance significantly exceeded the clearance of UPC-10. This wasexpected because treatment of cells with IFNγ increases surfaceexpression of HLA-A2 and the specific target, p68 peptide-HLA-A2.Further, the transendothelial clearance of RL-6A and BB7.2 wassignificantly decreased by competing unlabeled antibody.

Results from Example 2: The brain uptake of TCRm RL-6A is demonstratedin FIGS. 4 a-4 d and 5 a-5 d after intravenous bolus administration ofradiolabeled antibody to HLA-A2 transgenic mice of strain 3475 and 4191,respectively. In 3475 mice UPC-10 was used as negative control,representing brain plasma volume. After IFNγ, Vd of both RL-6A and BB7.2were significantly enhanced over control values (p<0.01 vs. UPC10, ANOVAwith Dunnett's post test). Co-injection of an excess (˜70 fold) ofunlabeled RL-6A decreases Vd brain to the background level (FIG. 4 b).The resulting brain concentrations of RL-6A and BB7.2 tracers aftercorrection for vascular space are shown in (FIG. 4 c). The result ofcapillary depletion analysis for R-L6A brain uptake is depicted in (FIG.4 d). More than 50% of tracer present in brain tissue was found inpostvascular supernatant, i.e. beyond the BBB, at the sampling time of60 min.

It was evident that IFNγ treatment in these mice did not cause BBBleakage, as no increase in Vd of the IgG2a isotype control was observed.In this strain the comparison of RL-6A brain uptake with or without IFNγpretreatment also indicates that basal expression of the targetpeptide-MHC complex is low. Stimulation of MHC processing andpresentation results in significant brain accumulation of RL-6A(0.19±0.03% 1 D/g) and BB7.2 as shown in FIG. 4 c, with the majorfraction undergoing transport across the BBB as concluded from capillarydepletion (FIG. 4 d). The uptake was fully saturated by a dose of 2mg/kg RL6A (FIG. 4 b) further supporting the specificity of the bindingand transport mechanism. Similar data were obtained for the strain 4191,expressing a different human HLA-A2 construct.

FIGS. 5 a-5 d show the brain uptake expressed as apparent Vd at 30 μg/kginjected dose (FIG. 5 a) and the saturation at 2 mg/kg for both RL-6Aand BB7.2. FIG. 5 c depicts the brain concentrations after correctionfor vascular space (8.5 μL/g, value taken from 3475 mice). The RL-6Adata are given for hemi and homozygous mice, no statistical comparisonswere made at this point due to the small n.

FIGS. 6 a-6 b illustrate the kinetics in plasma obtained in thesestudies. At the low tracer doses (˜30 μg/kg) we observed rapid initialdecline of plasma concentrations already at the earliest sampling timecompared to UPC10 (FIG. 6 a, strain 3475), which indicates clearancefrom the circulation by binding to peptide-MHC complexes in the body. Atthe 2 mg/kg dose, plasma pharmacokinetics demonstrates saturation ofclearance, with plasma concentration time curves similar to the isotypecontrol antibody. To check for the integrity of the tracers in the invivo experiments, precipitation by TCA was used. All tracers showedprecipitability >98% before injection. Precipitability in plasma sampleswas typically 98-99% throughout the sampling period and >90% in brainsamples.

Making of TCR Mimics

The heavy and light chains of, e.g., a HLA-A2 Class I molecule areexpressed and prepared separately in E. coli as insoluble inclusionbodies according to established protocols, solubilized and isolated astetramers. Balb/c mice (female and male) are immunized with the HLA-A2tetramers. Each mouse was injected subcutaneously every 2 weeks (up to 5times) with immunogen (50.mu.g) in PBS which also contained 25 ug ofQuil A (adjuvant) in 100 ul. Blood samples from mice were collected into1.5 ml eppendorf microcentrifuge tubes containing heparin, and plasmawas clarified by centrifugation. A significant portion of the antibodiesraised against peptide-HLA tetramers are generated against HLA as wellas streptavidin (SA) utilized to tetramerize the peptide-HLA complexes;consequently, an assay protocol had to be developed that allowed fordetection of a low concentration of specific antibodies in a milieu ofnon-specific ones. To resolve this problem, a pre-absorption step wasincorporated into an ELISA assay format. In the ELISA format, sera fromimmunized mice are first reacted with HLA-A2 monomers containing anotherirrelevant peptide before reacting them with HLA-A2 complexes of therelevant peptide. Serum from the immunized mice was used in an ELISAformat to identify “peptide-specific” antibody responses. Remember thatTCR mimics are antibodies having dual specificity for both peptide andHLA. In addition, the immunized mice will produce antibody specificitiesagainst HLA epitopes. It is these antibodies that the pre-absorptionprotocol substantially removes from the serum samples. In order tosubstantially remove antibodies that were not peptide specific, apre-absorption step was included in the protocol.

Using HLA-A2 as an example, a positive control in the assay, BB7.2 mAbwas used at 50 to 200 ng/well. This mAb recognizes only conformationallycorrect forms of the refolded peptide-HLA-A2 molecule. For a negativecontrol in the assay, a peptide-HLA-A2 complex containing an irrelevantpeptide was coated on the plate. In this particular assay, the negativecontrol was eIF4G peptide-loaded HLA-A2 monomer.

Hybridomas were generated by submitting 12 mice immunized with264p-HLA-A2 to the Hybridoma are generated using standard technology.Supernatants are screened for hybridomas to determine if they areproducing anti-peptide-HLA specific antibodies. Hybridomas determinedpositive after a first screening re expanded, and the supernatant arediluted and rescreened by competitive ELISA. Anti-HLA-target pluspeptide specificity of TCRm's are validated for generating monoclonalantibodies specific for peptide-HLA complexes.

Differential Proapoptotic Effect of TCR Mimic Antibodies On BreastCancer And Brain Endothelial Cells

Monoclonal antibodies are widely used as immunotherapeutics in cancerbut they often lack specificity for tumor cells vs. normal cells, whichis one of their limitations. The search for tumor specific antigens ledto the development of TCR mimic antibodies (TCRm) that bind to thepeptides expressed on MHC class I. Two of these TCRm, RL6A (againstpeptide YLLPAIVHI-HLA A2 complex) and RL21A (against FLSELTQQL-HLA A2complex), have considerable antitumor activity against breast cancercells. RL6A is transported across the blood-brain barrier. As there iscurrently no effective therapy for brain metastasis of breast cancer,the extent to which the RL6A crosses the blood-brain-barrier wasdetermined. The efficacy of RLA6 to cross the blood-barin-barrier wascompared to other antibodies with respect to induction of apoptosis inthe brain-seeking subclone 231-BR of the triple negative breast cancercell line MDA-MB-231. In parallel, the inventors studied the effect onbrain endothelial cells.

Methods. MDA-MB-231 and MDA-MB-231 BR cell lines were treated with RL6A,RL21A and UPC10 (IgG2a isotype control) for 24 hours at 370 C in 96 wellplates. Similar treatment was given to human brain endothelial cells(cell line HH8 and hCMECD3). The activity of cleaved Caspase3 andcleaved PARP was determined by ELISA (Pierce) (See, Verma B, Jain R,Casseltines S, Rennels A, Bhattacharya R, Markiewski M M, Rawat A,Neethling F, Bickel U, Weidanz J. TCR mimic monoclonal antibodies induceapoptosis of tumor cells via immune effector-independet mechanisms(2011) J Immunol. 186 (5): 3265-76). The data were fitted by 4-parameterlogistic regression and curves were compared by F-test (GraphPad Prism6c).

It was found that, RL6A & RL21A showed higher cleaved Caspase 3 activitycompared to isotype control. Both antibodies also showed significantlyhigher cleaved PARP activity compared to UPC 10. RL6A & RL21A showednegligible cleaved Caspase3 and cleaved PARP activity in monolayeredcell lines (HH8, hCMED3). Both antibodies still retain some of theirapoptotic activities in growth phase of HH8 as well as the continuouslygrowing brain endothelial cells hCMECD3.

Thus, it can be concluded that RL6A is a potent apoptotic agent in 231BR comparable to parent MDA-MB-231 breast cancer cell lines.Importantly, it did not affect the monolayered brain endothelial celllines suggesting that the normal BBB may not be compromised while usingthis antibody against brain metastases. Finally, the RL6A antibody didshow some apoptotic effect on the growth phase of the endothelial cells,suggesting that it might show an apoptotic effect on the angiogenicendothelial cells that are seen at BTB.

FIG. 8 shows the internalization of RL6A in MDA-MB-231 cells. FIG. 9shows the expression of RL6A on cell surface of brain endothelial cellline. FIGS. 10A and 10B show the uptake of RL6A in brain endothelialcells.

FIGS. 11A and 11B shows the binding of the antibodies on MDA-MB-231cells. FIGS. 12A and 12B show the effect of the antibodies for cleavedCaspase 3 (12A) and cleaved PARP (12B), with hCMEDC3 cells in growthphase and monolayered. FIG. 13 shows that RL6A in MDA-MB-231 BR cellline showed significantly higher cleaved Caspase 3 activity compared tobrain endothelial cells.

FIGS. 14A and 14B shows the binding of the antibodies on MDA-MB-231cells. FIGS. 15A and 15B show the effect of the antibodies for cleavedCaspase 3 (12A) and cleaved PARP (12B), with HH8 cells in growth phaseand monolayered. FIG. 16 shows that cleaved PARP activity wassignificantly higher in RL6A treated MDA-MB-231 BR compared to brainendothelial cell lines.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

Bhattacharya R, Xu Y, Rahman M A, Couraud P O, Romero I A, Weksler B B,Weidanz J A, Bickel U. A novel vascular targeting strategy forbrain-derived endothelial cells using a TCR mimic antibody. J CellPhysiol 2010; 225: 664-672.

Lee H J, Engelhardt B, Lesley J, Bickel, Pardridge W M. Targeting ratanti-mouse transferrin receptor monoclonal antibodies throughblood-brain barrier in mouse. J Pharmacol Exp Ther 2000; 292: 1048-1052.

Neethling F A, Ramakrishna V, Keler T, Buchli R, Woodburn T, Weidanz JA. Assessing vaccine potency using TCR mimic antibodies. Vaccine 2008;26: 3092-3102.

Verma B, Hawkins O E, Neethling F A, Caseltine S L, Largo S R,Hildebrand W H, Weidanz J A. Direct discovery and validation of apeptide/MHC epitope expressed in primary human breast cancer cells usinga TCRm monoclonal antibody with profound antitumor properties. CancerImmunol Immunother 201 0; 59: 563-573.

Verma B, Neethling F A, Caseltine S, Fabrizio G, Largo S, Duty J A,Tabaczewski P, Weidanz J A. TCR mimic monoclonal antibody targets aspecific peptide/HLA class I complex and significantly impedes tumorgrowth in vivo using breast cancer models. J Immunol 2010; 184:2156-2165.

Weidanz J A, Nguyen T, Woodburn T, Neethling F A, Chiriva-Internati M,Hildebrand W H, Lustgarten J. Levels of specific peptide-HLA class Icomplex predicts tumor cell susceptibility to CTL killing. JImmuno/2006; 177: 5088-5097.

Weidanz J A, Piazza P, Hickman-Miller H, Woodburn D, Nguyen T, Wahl A,t¥Jeethling F, Chiriva-Internati M, Rinaldo C R, Hildebrand W H.Development and implementation of a direct detection, quantitation andvalidation system for class I MHC self-peptide epitopes. J ImmunolMethods 2007; 318: 47-58.

Weksler B B, Subileau E A, Perriere N, Charneau P, Holloway K, LevequeM, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male D K,Roux F, Greenwood J, Romero I A, Couraud PO. Blood-brainbarrier-specific properties of a human adult brain endothelial cellline. Faseb J 2005; 19: 1872-1874.

What is claimed is:
 1. A method of delivering a therapeutic agent intoand across an endothelial cell (EC) in a subject in need thereof,comprising: identifying a subject in need for treatment of a cancer inthe brain; attaching to a TCR mimic an active agent to form atherapeutic agent; and administering to the subject the therapeuticagent in a pharmaceutically acceptable carrier, wherein the therapeuticagent effectively crosses the blood-central nervous system microvascularbarrier.
 2. The method of claim 1, wherein the barrier being crossed isthe blood-brain barrier (BBB), blood-retina barrier, blood-nerve barrieror blood-spinal cord barrier.
 3. The method of claim 1, wherein thedisease or disorder is a cancer other than a brain cancer that hasmetastasized to the brain.
 4. The method of claim 1, wherein the diseaseor disorder is a breast cancer that has metastasized to the brain. 5.The method of claim 1, wherein the TCR mimic is an antibody or fragmentthereof and is selected from RL6A or RL21A.
 6. The method of claim 1,wherein the therapeutic agent or carrier is directly conjugated to atargeting molecule by: (a) non-specific or specific protein-proteininteraction; (b) covalent bonding; (c) non-covalent bonding; or (d)coordinating chemical bonding; which conjugation is optionally effectedvia a spacer or linker that bridges between the therapeutic agent orcarrier and the targeting molecule, or (e) a recombinant fusion orhybrid polypeptide.
 7. The method of claim 1, wherein the step ofadministering is (a) by continuous intravenous or intraarterialinfusion; or (b) by bolus injection by an intravenous, intramuscular,intraarterial, or intralesional route.
 8. The method of claim 1, whereinthe active agent is an antineoplastic agent, a cytotoxic agent,anti-inflammatory, a hormone, an enzyme, a neurotransmitter, aneurotrophic factor, antibiotics, a cytokine, or a neuropeptide.
 9. Amethod of delivering a therapeutic agent into and across a microvascularblood-central nervous system (CNS) barrier in a subject, comprising:identifying a subject in need for treatment of a cancer in the brain;and administering to a subject with at least one of a CNS disease ordisorder or a peripheral disease or disorder with CNS involvement, acomposition comprising: a TCR mimic and active agent that together forma therapeutic agent; and a pharmaceutically acceptable carrier, whereinthe therapeutic agent is encapsulated in a nanocontainer to which islinked the targeting molecule, wherein an effective barrier-entering andbarrier crossing amount of the therapeutic agent enters and crosses themicrovascular blood CNS barrier.
 10. The method of claim 9, wherein thebarrier being crossed is the blood-brain barrier (BBB), blood-retinabarrier, blood-nerve barrier or blood-spinal cord barrier.
 11. Themethod of claim 9, wherein the disease or disorder is a cancer otherthan a brain cancer that has metastasized to the brain.
 12. The methodof claim 9, wherein the disease or disorder is a breast cancer that hasmetastasized to the brain.
 13. The method of claim 9, wherein the TCRmimic is an antibody or fragment thereof and is selected from RL6A orRL21A.
 14. The method of claim 9, wherein the therapeutic agent orcarrier is directly conjugated to a targeting molecule by: (a)non-specific or specific protein-protein interaction; (b) covalentbonding; (c) non-covalent bonding; or (d) coordinating chemical bonding;which conjugation is optionally effected via a spacer or linker thatbridges between the therapeutic agent or carrier and the targetingmolecule, or (e) a recombinant fusion or hybrid polypeptide.
 15. Themethod of claim 9, wherein the step of administering is at least one of:(a) by continuous intravenous or intraarterial infusion; or (b) by bolusinjection by an intravenous, intramuscular, intraarterial, orintralesional route.
 16. The method of claim 9, wherein the active agentis an antineoplastic agent, a cytotoxic agent, anti-inflammatory, ahormone, an enzyme, a neurotransmitter, a neurotrophic factor,antibiotics, a cytokine, or a neuropeptide.
 17. A method for treating aneuronal disease or condition comprising: identifying a subject in needof such treatment for a cancer in the brain; administering to thesubject in need of such treatment a neuroprotective and/orneurorestorative agent in an amount effective to treat the disorder inthe subject, wherein the agent comprises: a TCR mimic that targets anMHC-peptide combination found on the surface of an endothelial cells ofthe neuronal vasculature that is conjugated to an active agent to form atherapeutic agent and that crosses at least one of the blood-brainbarrier, blood-retina barrier, blood-nerve barrier, or blood-spinal cordbarrier.
 18. The method of claim 17, wherein the disease or disorder isa cancer other than a brain cancer that has metastasized to the brain.19. The method of claim 17, wherein the disease or disorder is a breastcancer that has metastasized to the brain.
 20. The method of claim 17,wherein the TCR mimic is an antibody or fragment thereof and is selectedfrom RL6A or RL21A.
 21. The method of claim 17, wherein the therapeuticagent or carrier is directly conjugated to a targeting molecule by: (a)non-specific or specific protein-protein interaction; (b) covalentbonding; (c) non-covalent bonding; or (d) coordinating chemical bonding;which conjugation is optionally effected via a spacer or linker thatbridges between the therapeutic agent or carrier and the targetingmolecule, or (e) a recombinant fusion or hybrid polypeptide.
 22. Themethod of claim 17, wherein the step of administering is at least one of(a) by continuous intravenous or intraarterial infusion; or (b) by bolusinjection by an intravenous, intramuscular, intraarterial, orintralesional route.
 23. The method of claim 17, wherein the activeagent is an antineoplastic agent, a cytotoxic agent, anti-inflammatory,a hormone, an enzyme, a neurotransmitter, a neurotrophic factor,antibiotics, a cytokine, or a neuropeptide.